A major endeavor in molecular genetics has been made in generating maps of the human genome. Human genome mapping consists, generally, of ordering genomic DNA fragments on their chromosomes using several methods, such as fluorescent in situ hybridization (FISH), somatic cell hybrid analysis or random clone fingerprinting. DNA fragments that correspond to marked polymorphic sites can be ordered by genetic linkage analysis. Distances between polymorphic loci are estimated by meiotic recombination frequencies. High resolution maps based upon the estimated distances, however, cannot be constructed easily using such methods because the resolution is low at the molecular level and recombination frequency is not linearly correlated with physical distance.
Thus, various obstacles such as, for example, the difficulty in obtaining highly informative markers and the paucity of identified markers that are evenly spaced along the chromosome are significant weaknesses of the currently available genetic maps. Most of the mapped markers are restriction fragment length polymorphisms (RFPLs) assayed by DNA hybridization. Although maps based on these markers have contributed greatly to the primary mapping of a number of diseases, they are still insufficient for many applications such as mapping rare monofactorial diseases, refining linkage intervals to distances suited for gene identification, and mapping of loci contributing to complex traits.
Genetic linkage mapping is an important technology applied to the study of human biology and, in particular, for the delineation of the molecular basis of disease. Indeed, one of the most commonly used strategies for studying human inherited diseases is by cloning the responsible gene based on chromosomal location. Genetic linkage maps, therefore, facilitate the identification and mapping of genes involved in monogenic diseases, genes involved in multifactorial disorders, and are useful in carrier detection and prenatal diagnosis of hereditary disorders. A detailed linkage map is also a prerequisite for clone-based physical mapping and DNA sequencing of the entire chromosome.
Human chromosome 21 is a paradigm for large-scale human genome mapping efforts. The smallest human chromosome, chromosome 21 has approximately 50 megabases (Mb) of DNA. Less than 1% of the 2000 genes estimated to be on chromosome 21 are known. A high resolution map of chromosome is of particular interest because of its apparent role in familial Alzheimer disease (FAD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), and Finnish progressive myocionus epilepsy (PME). A gene defect responsible for FAD has been localized to chromosome 21 on the basis of genetic linkage to three pericentromeric loci. The gene encoding the precursor of the Alzheimer-associated amyloid .beta. protein (APP), the principle component of the senile plaques and cerebrovascular amyloid deposits of Alzheimer disease (AD), has also been mapped to chromosome 21.
The process of developing such a long-range contig map involves the identification and localization of landmarks in cloned genetic fragments. When there are enough landmarks for the size of the cloned fragments, contigs are formed, and the landmarks are simultaneously ordered. Currently, YACS, or yeast artificial chromosomes, are utilized for most mapping of the human genome. YACs permit cloning of fragments of a about 500 Kb. However, some difficulties have been encountered with the manipulation of YAC libraries. For example, in various YAC libraries, a fraction of the clones result from co-cloning events, i.e., they include in a single clone noncontiguous DNA fragments. A high percentage of YAC clones, particularly clones having high molecular weight inserts, are chimeric. Chimeric clones map to multiple sites on the chromosome and, thus, hamper the progress of mapping and analysis. Another problem endemic to YAC cloning is caused by DNA segments that are unclonable or unstable and tend to rearrange and delete.
Bacteria Artificial Chromosomes (BACs), provide an alternative to the YAC system. BACs mitigate the most problematic aspects of YACs such as, for example the high rate of chimerism and clonal instability. BACs are based on the E. coli single-copy plasmid F factor and are capable of faithful propagation of DNA fragments greater than about 300 Kb in size. BACs have a number of physical properties that make them amenable to physical mapping, including easy manipulation and an absence of chimerism. The lack of chimerism and the capacity to propagate large exogenous insert DNAs make the BACs excellent candidates for chromosome walking and the generation of contiguous physical maps.
The need for molecular description of chromosome 21 derives directly from the association with several human genetic diseases. A map of contiguous units (contigs) covering this chromosome will speed the identification of the cause of these diseases. Indeed, a detailed map would provide immediate access to the genomic segment, including any pathological locus, as soon as it has been localized by genetic linkage or cytogenetic analysis.
Thus, a need exists for identifying, characterizing, and mapping the numerous genes that occupy loci on chromosome 21, which will expedite the rapid translation of high resolution chromosome maps into biological, medical and diagnostic applications. The present invention satisfies this need and provides related advantages as well.